Protocol to electroporate DNA plasmids into Ciona robusta embryos at the 1-cell stage.

Electroporation is a technique to introduce DNA constructs into cells using electric current. Here, we present a protocol to electroporate DNA plasmids into Ciona robusta embryos at the 1-cell stage. We describe steps for setting up and conducting electroporation. We then detail procedures for collecting, fixing, and mounting embryos and counting expression. This protocol can be used to study the expression of enhancers via reporter assays, manipulating cells using genes or modified genes such as dominant negatives, and genome editing. For complete details on the use and execution of this protocol, please refer to Song, et al.1.

Affinity-optimizing enhancer variants disrupt development.

Enhancers control the location and timing of gene expression and contain the majority of variants associated with disease1-3. The ZRS is arguably the most well-studied vertebrate enhancer and mediates the expression of Shh in the developing limb4. Thirty-one human single-nucleotide variants (SNVs) within the ZRS are associated with polydactyly4-6. However, how this enhancer encodes tissue-specific activity, and the mechanisms by which SNVs alter the number of digits, are poorly understood. Here we show that the ETS sites within the ZRS are low affinity, and identify a functional ETS site, ETS-A, with extremely low affinity. Two human SNVs and a synthetic variant optimize the binding affinity of ETS-A subtly from 15% to around 25% relative to the strongest ETS binding sequence, and cause polydactyly with the same penetrance and severity. A greater increase in affinity results in phenotypes that are more penetrant and more severe. Affinity-optimizing SNVs in other ETS sites in the ZRS, as well as in ETS, interferon regulatory factor (IRF), HOX and activator protein 1 (AP-1) sites within a wide variety of enhancers, cause gain-of-function gene expression. The prevalence of binding sites with suboptimal affinity in enhancers creates a vulnerability in genomes whereby SNVs that optimize affinity, even slightly, can be pathogenic. Searching for affinity-optimizing SNVs in genomes could provide a mechanistic approach to identify causal variants that underlie enhanceropathies.

Conserved enhancers control notochord expression of vertebrate Brachyury.

The cell type-specific expression of key transcription factors is central to development and disease. Brachyury/T/TBXT is a major transcription factor for gastrulation, tailbud patterning, and notochord formation; however, how its expression is controlled in the mammalian notochord has remained elusive. Here, we identify the complement of notochord-specific enhancers in the mammalian Brachyury/T/TBXT gene. Using transgenic assays in zebrafish, axolotl, and mouse, we discover three conserved Brachyury-controlling notochord enhancers, T3, C, and I, in human, mouse, and marsupial genomes. Acting as Brachyury-responsive, auto-regulatory shadow enhancers, in cis deletion of all three enhancers in mouse abolishes Brachyury/T/Tbxt expression selectively in the notochord, causing specific trunk and neural tube defects without gastrulation or tailbud defects. The three Brachyury-driving notochord enhancers are conserved beyond mammals in the brachyury/tbxtb loci of fishes, dating their origin to the last common ancestor of jawed vertebrates. Our data define the vertebrate enhancers for Brachyury/T/TBXTB notochord expression through an auto-regulatory mechanism that conveys robustness and adaptability as ancient basis for axis development.

Single-nucleotide variants within heart enhancers increase binding affinity and disrupt heart development.

Transcriptional enhancers direct precise gene expression patterns during development and harbor the majority of variants associated with phenotypic diversity, evolutionary adaptations, and disease. Pinpointing which enhancer variants contribute to changes in gene expression and phenotypes is a major challenge. Here, we find that suboptimal or low-affinity binding sites are necessary for precise gene expression during heart development. Single-nucleotide variants (SNVs) can optimize the affinity of ETS binding sites, causing gain-of-function (GOF) gene expression, cell migration defects, and phenotypes as severe as extra beating hearts in the marine chordate Ciona robusta. In human induced pluripotent stem cell (iPSC)-derived cardiomyocytes, a SNV within a human GATA4 enhancer increases ETS binding affinity and causes GOF enhancer activity. The prevalence of suboptimal-affinity sites within enhancers creates a vulnerability whereby affinity-optimizing SNVs can lead to GOF gene expression, changes in cellular identity, and organismal-level phenotypes that could contribute to the evolution of novel traits or diseases.

Conserved enhancer logic controls the notochord expression of vertebrate Brachyury.

The cell type-specific expression of key transcription factors is central to development. Brachyury/T/TBXT is a major transcription factor for gastrulation, tailbud patterning, and notochord formation; however, how its expression is controlled in the mammalian notochord has remained elusive. Here, we identify the complement of notochord-specific enhancers in the mammalian Brachyury/T/TBXT gene. Using transgenic assays in zebrafish, axolotl, and mouse, we discover three Brachyury-controlling notochord enhancers T3, C, and I in human, mouse, and marsupial genomes. Acting as Brachyury-responsive, auto-regulatory shadow enhancers, deletion of all three enhancers in mouse abolishes Brachyury/T expression selectively in the notochord, causing specific trunk and neural tube defects without gastrulation or tailbud defects. Sequence and functional conservation of Brachyury-driving notochord enhancers with the brachyury/tbxtb loci from diverse lineages of fishes dates their origin to the last common ancestor of jawed vertebrates. Our data define the enhancers for Brachyury/T/TBXTB notochord expression as ancient mechanism in axis development.

3D genomics across the tree of life reveals condensin II as a determinant of architecture type.

We investigated genome folding across the eukaryotic tree of life. We find two types of three-dimensional (3D) genome architectures at the chromosome scale. Each type appears and disappears repeatedly during eukaryotic evolution. The type of genome architecture that an organism exhibits correlates with the absence of condensin II subunits. Moreover, condensin II depletion converts the architecture of the human genome to a state resembling that seen in organisms such as fungi or mosquitoes. In this state, centromeres cluster together at nucleoli, and heterochromatin domains merge. We propose a physical model in which lengthwise compaction of chromosomes by condensin II during mitosis determines chromosome-scale genome architecture, with effects that are retained during the subsequent interphase. This mechanism likely has been conserved since the last common ancestor of all eukaryotes.

Real-time interactive isosurfacing: a new method for improving marching isosurfacing algorithm output and efficiency.

Efficient rendering of a changing volumetric data-set is central to the development of effective medical simulations that incorporate haptic feedback. A new method referred to as real-time interactive isosurfacing (RTII) is described in this paper. RTII is an algorithm that can be applied to output from Marching Cubes-like algorithms to improve performance for real-time applications. The approach minimises processing by re-evaluating the isosurface around changing sub-volumes resulting from user interactions. It includes innovations that significantly reduce mesh complexity and improve mesh quality as triangles are created from the Marching Tetrahedra isosurfacing algorithm. Rendering efficiency is further improved over other marching isosurfacing algorithm outputs by maintaining an indexed triangle representation of the mesh. The effectiveness of RTII is discussed within the context of an endoscopic sinus surgery simulation currently being developed by the authors.

Usability of tablet computers by people with early-stage dementia.

BACKGROUND: Tablet computers are generally associated with an intuitive interface. The adoption and use of tablet computers within the early-stage dementia context could potentially assist in daily living and provide users with a source for leisure activities and social networking. As dementia mainly affects the older adult population, it is expected that many people with dementia and even their carers do not use tablet computers as part of their everyday living. OBJECTIVE: This paper explores the usability of tablet computers within the early-stage dementia context as a source of leisure for people with dementia. The main advantage of the use of tablet computers in this manner is to provide carers some reprieve from the constant care and attention often required in caring for people with dementia. METHODS: Seven-day in-home trials were conducted to determine whether people with early-stage dementia were -capable of using a tablet computer independently. Twenty-one people with early-stage dementia and carer dyads participated in the trial. Feedback was gathered through questionnaires from both the person with dementia and their carer regarding the use of a tablet computer as part of their everyday living. RESULTS: Approximately half the participants with dementia were able to engage with and use the tablet computer independently, which proved to be helpful to their carers. No significant traits were observed to help identify those who were less likely to use a tablet computer. Carer relief was quantified by the amount of time participants with dementia spent using the device without supervision. CONCLUSIONS: The results and feedback from the trial provide significant insights to introducing new technology within the early-stage dementia context. Users' needs must be considered on a case-by-case basis to successfully facilitate the uptake of tablet computers in the dementia context. The trial has provided sufficient justification to further explore more uses of tablet computers in the dementia context, and not just for early-stage dementia.

A prospective cohort study on the impact of a modified Basic Military Training (mBMT) programme based on pre-enlistment fitness stratification amongst Asian military enlistees.

INTRODUCTION: This study objectively evaluates the effectiveness of a 6-week Preparatory Training Phase (PTP) programme prior to Basic Military Training (BMT) for less physically conditioned conscripts in the Singapore Armed Forces. MATERIALS AND METHODS: We compared exercise test results of a group of less fi t recruits who underwent a 16-week modified-BMT (mBMT) programme (consisting of a 6-week PTP and 10-week BMT phase) with their 'fitter' counterparts enlisted in the traditional 10-week direct-intake BMT (dBMT) programme in this prospective cohort study consisting of 36 subjects. The main outcome measures included cardiopulmonary responses parameters (VO(2)max and V(O2AT)) with clinical exercise testing and distance run timings. RESULTS: Although starting off at a lower baseline in terms of physical fitness [VO(2)max 1.73 +/- 0.27 L/min (mBMT group) vs 1.97 +/- 0.43 L/min (dBMT), P = 0.032; V(O2AT) 1.02 +/- 0.19 vs 1.14 +/- 0.32 L/min respectively, P = 0.147], the mBMT group had greater improvement in cardiopulmonary indices and physical performance profiles than the dBMT cohort as determined by cardiopulmonary exercise testing [VO(2)max 2.34 +/- 0.24 (mBMT) vs 2.36 +/- 0.36 L/min (dBMT), P = 0.085; V(O2AT) 1.22 +/- 0.17 vs 1.21 +/- 0.24 L/min respectively, P = 0.303] and 2.4 kilometres timed-run [mBMT group 816.1 sec (pre-BMT) vs 611.1 sec (post-BMT), dBMT group 703.8 sec vs 577.7 sec, respectively; overall P value 0.613] at the end of the training period. Initial mean difference in fitness between mBMT and dBMT groups on enlistment was negated upon graduation from BMT. CONCLUSION: Pre-enlistment fitness stratification with training modification in a progressive albeit longer BMT programme for less-conditioned conscripts appears efficacious when measured by resultant physical fitness.

Instrument-tissue segment interaction using finite element modeling.

A virtual reality based laparoscopic surgery simulator is an important training option for laparoscopic surgeons. It has significant advantages over other training methods. Instruments-anatomy interactions are one of the main features of these simulators. In this paper we present the deformation of the uterine tube using three dimensional finite element methods with finite element software. The work examines the feasibility of incorporating the finite element (FE) model within the visual graphic model to achieve high degree of realism of instrument-tissue interactions.

A visual graphic/haptic rendering model for hysteroscopic procedures.

Hysteroscopy is an extensively popular option in evaluating and treating women with infertility. The procedure utilises an endoscope, inserted through the vagina and cervix to examine the intra-uterine cavity via a monitor. The difficulty of hysteroscopy from the surgeon's perspective is the visual spatial perception of interpreting 3D images on a 2D monitor, and the associated psychomotor skills in overcoming the fulcrum-effect. Despite the widespread use of this procedure, current qualified hysteroscopy surgeons have not been trained the fundamentals through an organised curriculum. The emergence of virtual reality as an educational tool for this procedure, and for other endoscopic procedures, has undoubtedly raised interests. The ultimate objective is for the inclusion of virtual reality training as a mandatory component for gynaecologic endoscopy training. Part of this process involves the design of a simulator, encompassing the technical difficulties and complications associated with the procedure. The proposed research examines fundamental hysteroscopy factors, current training and accreditation, and proposes a hysteroscopic simulator design that is suitable for educating and training.

Haptic rendering for VR laparoscopic surgery simulation.

This project concerns the application of haptic feedback to a VR laparoscopic surgery simulator. Haptic attributes such as mass, friction, elasticity, roughness and viscosity are individually modeled, validated and applied to the existing visual simulation created by researchers at Monash University. Haptic feedback is an essential element in an immersive and realistic virtual reality laparoscopic training simulator. The haptic system must display stable, continuous and realistic multi-dimensional force feedback, and its inclusion should enhance the simulators training capability. Stability is a recurring concern throughout haptic history, and will be tackled with the implementation of a stable control algorithm and a passive environment model. Haptic force feedback modeling, systems implementation and validation studies form the principal areas of new work associated with this project.

Hysteroscopic simulator for training and educational purposes.

Hysteroscopy is an extensively popular option in evaluating and treating women with infertility. The procedure utilizes an endoscope, inserted through the vagina and cervix to examine the intra-uterine cavity via a monitor. The difficulty of hysteroscopy from the surgeon's perspective is the visual spatial perception of interpreting 3D images on a 2D monitor, and the associated psychomotor skills in overcoming the fulcrum-effect. Despite the widespread use of this procedure, current qualified hysteroscopy surgeons have not been trained the fundamentals through an organized curriculum. The emergence of virtual reality as an educational tool for this procedure, and for other endoscopic procedures, has undoubtedly raised interests. The ultimate objective is for the inclusion of virtual reality training as a mandatory component for gynecological endoscopic training. Part of this process involves the design of a simulator, encompassing the technical difficulties and complications associated with the procedure. The proposed research examines fundamental hysteroscopic factors as well as current training and accreditation norms, and proposes a hysteroscopic simulator design that is suitable for educating and training.

Haptic rendering & perception studies for laparoscopic surgery simulation.

This project concerns the application of haptic feedback to a virtual reality laparoscopic surgery simulator. Haptic attributes such as mass, friction, stiction, elasticity, roughness and viscosity are individually modeled, validated and applied to the existing visual simulation created by researchers at Monash University. Validation studies has shown that refinements to our mechanical interface improves the accuracy of localisation by 25%. Using our mechanical interface, the JND (Just Noticeable Difference) for instantaneous change of magnitude of haptic attributes is approximately 12%. This suggests the mechanical interface is suitable to use for surgery based studies. There are times in surgery when the view from the camera cannot be depended upon. When visual feedback is impeded, haptic feedback must be relied upon more by the surgeon. A realistic simulator should include some sort of visual impedance. Results from a simple tissue holding task suggested the inclusion of haptic feedback in a simulator aids the user when visual feedback is impeded. Haptic force feedback modeling, systems implementation, threshold and level perception, and validation studies form the principal areas of new work associated with this project.